The unavailability of acceptable human donor organs, the low rate of long term success due to host versus graft rejection, and the serious risks of infection and cancer are the main challenges now facing the field of tissue and organ transplantation. Because the demand for acceptable organs exceeds the supply, many people die each year while waiting for organs to become available. To help meet this demand, research has been focused on developing alternatives to allogenic transplantation. For example, dialysis has been available to patients suffering from kidney failure, artificial heart models have been tested, and other mechanical systems have been developed to assist or replace failing organs. Such approaches, however, are quite expensive, and the need for frequent and periodic access to machines greatly limits the freedom and quality of life of patients undergoing this type of therapy.
Xenograft transplantation represents a potentially attractive alternative to artificial organs for human transplantation. The potential pool of nonhuman organs is virtually limitless, and successful xenograft transplantation would not render the patient virtually tethered to machines as is the case with artificial organ technology. Host rejection of such cross-species tissue, however, remains a major hurdle in this area, and the success of organ transplants depends on avoiding rejection of the transplant.
The forms of transplant rejection are clinically classified by their time frames and histologies. Hyperacute rejection (HAR), for example, occurs within minutes to hours following transplant. Hyperacute rejection is characterized by rapid thrombotic occlusion of the graft vasculature that begins within minutes to hours after host blood vessels are anastomosed to graft vessels. Hyperacute rejection is mediated by antibodies that pre-exist in naive hosts, the so-called ‘natural antibodies,’ which bind to endothelium and activate complement. Antibody and complement induce a number of changes in the graft endothelium that promote intravascular thrombosis. On the other hand, acute rejection typically occurs within 1-30 days, and chronic rejection occurs thereafter, sometimes taking several months to years. Some noted xenotransplants of organs from apes or old-world monkeys (e.g., baboons) into humans have been tolerated for months without rejection. However, such attempts have ultimately failed due to a number of immunological factors. Even with heavy immunosuppressive drugs used to suppress HAR, a low-grade innate immune response ultimately leads to destruction of the transplanted organs. This low grade innate immune response is attributable, in part, to failure of complement regulatory proteins (CRPs) within the graft tissue to control activation of heterologous complement on graft endothelium (see e.g., Starzl et al., Immunol. Rev., 141, 213-44 (1994)). In addition to HAR, DXR, also known as acute vascular rejection, and T-cell mediated responses also play a major role in host graft rejection. It is likely that a multifaceted strategy will need to be employed to overcome the barriers to successfully transplant non-human organs into human recipients.
Complicating the efficacy of xenotransplants further is the fact that drugs used to control innate immune responses to the xenograft can cause a non-specific depression of the immune system. Patients on such immune suppressive agents are more susceptible to the development of life-threatening infections and neoplasia.
In an effort to develop a pool of immuno-acceptable organs for xenotransplantation into humans, researchers have engineered animals producing human CRPs, an approach which has been demonstrated to delay, but not eliminate, xenograft destruction in primates (McCurry et al., Nat. Med., 1, 423-27 (1995); Bach et al., Immunol. Today, 17, 379-84 (1996)). However, organs surviving HAR may still be subjected to delayed xenograft rejection (DXR). This is characterized by the infiltration of recipient inflammatory cells and thrombosis of graft vessels, leading to ischaemia of the organ.
Whereas HAR is associated with rapid, protein-synthesis-independent, type I endothelial cell activation that results in graft rejection within minutes or hours, DXR, also known as acute vascular rejection, relates primarily to type II endothelial cell activation (see Bach F. H. et al., Immunology Today 17(8):379-384 (1996)). This response involves transcriptional induction of genes and subsequent protein synthesis resulting in the expression of adhesion molecules, cytokines, procoagulant molecules and others (Prober J. S. et al., Transplantation 50: 537-544 (1990); Prober J. S. et al., Physiol. Rev. 70: 427451 (1990); Cotran R. S. et al., Kidney Ins. 35: 969-975 (1989)). DXR is characterized by the infiltration into the graft of host monocytes and natural killer cells (NK), which promote intragraft inflammation and thrombosis (Bach F. H. et al., Immunology Today 17(8):379-384 (1996)).
Inhibition of complement by soluble complement receptor type I (sCR1) combined with immunosuppression has been reported to delay the occurrence of DXR/AVR of porcine hearts transplanted into cynomoigus monkeys (Davis, EA et al., Transplantation 62:1018-23 (1996)). Transplantation of pig kidneys expressing human decay accelerating factor to cynomoigus monkeys also had some protective effect against DXR/AVR (Zaid A. et al., Transplantation 65:1584-90 (1998); Loss M et al., Xenotransplantation 7. 186.9 (2000)).
PCT Publication WO 02/30985A2 to Tanox Inc., teaches a method to suppress E-selectin in order to reduce DXR responses. E-selectin (also known as ELAM-1, CD62, and CD62E) is a cytokine inducible cell surface glycoprotein cell adhesion molecule that is found exclusively on endothelial cells. E-selectin mediates the adhesion of various leukocytes, including neutrophils, monocytes, eosinophils, natural killer (NK) cells, and a subset of T cells, to activated endothelium (Bevilacqua, et al., Science 243: 1160 (1989); Shimuzu, et al., Nature 349:799 (1991); Graber, et al., J. Immunol. 145: 819 (1990); Carlos, et al., Blood 77: 2266 (1991); Hakkert, et al., Blood 78:2721 (1991); and Picker, et al., Nature 349:796 (1991)). The expression of E-selectin is induced on human endothelium in response to the cytokines IL-1 and TNF, as well as bacterial lipopolysaccharide (LPS), through transcriptional up-regulation (Montgomery, et al., Proc Natl Acad Sci 88:6523 (1991)). The human leukocyte receptor for human E-selectin has been identified (Berg, et al., J. Biol. Chem. 23: 14869 (1991) and Tyrrell, et al., Proc Natl Acad Sci 88:10372 (1991)). Structurally, E-selectin belongs to a family of adhesion molecules termed “selectins” that also includes P-selectin and L-selectin (see reviews in Lasky, Science 258:964 (1992) and Bevilacqua and Nelson, J. Clin. Invest. 91:379 (1993)). These molecules are characterized by common structural features such as an amino-terminal lectin-like domain, an epidermal growth factor (EGF) domain, and a discrete number of complement repeat modules (approximately 60 amino acids each) similar to those found in certain complement binding proteins. Clinically, increased E-selectin expression on endothelium is associated with a variety of acute and chronic leukocyte-mediated inflammatory reactions including allograft rejection (Allen, et al., Circulation 88: 243 (1993); Brockmeyer, et al., Transplantation 55:610 (1993); Ferran, et al Transplantation 55:605 (1993); and Taylor, et al., Transplantation 54: 451 (1992)). Studies in which the expression of human E-selectin in cardiac and renal allografts undergoing acute cellular rejection was investigated have demonstrated that E-selectin expression is selectively up-regulated in vascular endothelium of renal and cardiac tissue during acute rejection (Taylor, et al., Transplantation 54: 451 (1992)). Additionally, increased E-selectin expression correlates with the early course of cellular rejection and corresponds to the migration of inflammatory cells into the graft tissue (Taylor, et al., Transplantation 54: 451 (1992)). Taken together, these studies provide evidence that cytokine-induced expression of E-selectin by donor organ endothelium contributes to the binding and subsequent transmigration of inflammatory cells into the graft tissue and thereby plays an important role in acute cellular allograft rejection.
In addition to complement-mediated attack, human rejection of discordant xenografts appears to be mediated by a common antigen: the galactose-α(1,3)-galactose (gal-α-gal) terminal residue of many glycoproteins and glycolipids (Galili et al., Proc. Nat. Acad. Sci., (USA), 84, 1369-73 (1987); Cooper, et al., Immunol. Rev., 141, 31-58 (1994); Galili, et al., Springer Sem. Immunopathol, 15, 155-171 (1993); Sandrin, et al., Transplant Rev., 8, 134 (1994)). This antigen is chemically related to the human A, B, and O blood antigens, and it is present on many parasites and infectious agents, such as bacteria and viruses. Most mammalian tissue also contains this antigen, with the notable exception of old world monkeys, apes and humans. (Joziasse, et al., J. Biol. Chem., 264, 14290-97 (1989)). Individuals without such carbohydrate epitopes produce abundant naturally occurring antibodies (IgM as well as IgG) specific to the epitopes. Many humans show significant levels of circulating IgG with specificity for gal-α-gal carbohydrate determinants (Galili, et al., J. Exp. Med, 162, 573-82 (1985); Galili, et al., Proc. Nat. Acad Sci. (USA), 84, 1369-73 (1987)). The α-galactosyltransferase (α-GT) enzyme catalyzes the formation of gal-α-gal moieties. Research has focused on the modulation or elimination of this enzyme to reduce or eliminate the expression of gal-α-gal moieties on the cell surface of xenotissue.
The elimination of the α-galactosyltransferase gene from porcine has long been considered one of the most significant hurdles to accomplishing xenotransplantation from pigs to humans. Two alleles in the pig genome encode the α-GT gene. Single allelic knockouts of the α-GT gene in pigs were reported in 2002 (Dai, et al. Nature Biotechnol., 20:251 (2002); Lai, et al., Science, 295:1089 (2002)).
Recently, double allelic knockouts of the α-GT gene have been accomplished (Phelps, et al., Science, 299: pp. 411-414 (2003)). WO 2004/028243 to Revivicor Inc. describes porcine animal, tissue, organ, cells and cell lines, which lack all expression of functional α1,3 galactosyltransferase (α1,3-GT). Accordingly, the animals, tissues, organs and cells lacking functional expression of α1,3-GT can be used in xenotransplantation and for other medical purposes.
PCT patent application WO 2004/016742 to Immerge Biotherapeutics, Inc. describes α(1,3)-galactosyltransferase null cells, methods of selecting GGTA-1 null cells, α(1,3)-galactosyltransferase null swine produced therefrom (referred to as a viable GGTA-1 null swine), methods for making such swine, and methods of using cells, tissues and organs of such a null swine for xenotransplantation.
α(1,3)-Galactosyltransferase, however, is not the only enzyme that synthesizes the Galα(1,3)Gal motif. Originally, Galα(1,3)Gal was thought to be exclusively synthesized by α(1,3)GT. More recent studies show that isogloboside 3 (iGb3) synthase is also capable of synthesizing Galα(1,3)Gal motifs (Taylor SG, et al Glycobiology 13(5): 327-337 (2003)). In contrast to α(1,3)GT, iGb3 synthase preferentially modifies glycolipids over glycoprotein substrates (Keusch et al. (2000) J.Bio.Chem. 275:25308-25314). iGb3 synthase acts on lactosylceramide (LacCer (Galβ1,4Glcβ1Cer)) to form the glycolipid isogloboid structure iGb3 (Galα1,3Galβ1,4Glcβ1Cer), initiating the synthesis of the isoglobo-series of glycoshingolipids.
Studies performed on rats confirm that two independent genes encoding distinct glycosyltransferases, α(1,3)GT and iGb3 synthase, are capable of synthesizing the Galα(1,3)Gal motif (Taylor et al. (2003) Glycobiology 13(5):327-337). These separate and distinct glycosyltransferases act through two different glycosylation pathways. Transfection studies have shown that α(1,3)GT synthesizes Galα(1,3)Gal on glycoproteins, whereas the synthesis of the Galα(1,3)Gal motif on the glycolipid is facilitated by iGB3 synthase. In addition, it has been shown that α(1,3)GT is incapable of synthesizing the Galα(1,3)Gal on glycolipids (Taylor et al. (2003) Glycobiology 13(5):327-337). These findings have refuted the previously held belief that α(1,3)GT was the sole Galα(1,3)Gal motif synthesizing enzyme.
The presence of the iGb3 synthase gene, and its contribution to the biosynthesis of the highly immunogenic Galα(1,3)Gal epitope, presents an additional hurdle to overcome in the quest for the production of immuno-tolerable xenotransplants.
Keusch J J et al have previously reported the cloning of the rat iGb3 synthase gene (J.Biol. Chem 2000). The gene is reported as GenBank sequence NM 138524.
PCT publication No. WO 02/081688 to The Austin Research Institute discloses a partial cDNA sequence encoding a portion of exon 5 (480 base pairs) of the porcine iGb3 synthase gene. This application also purports to cover the use of this DNA sequence to disrupt this gene in cells, tissues and organs for xenotransplantation.
It is an object of the present invention to provide genomic and regulatory sequences of the porcine iGb3 synthase gene.
It is an additional object of the present invention to provide cDNA sequences, as well as novel variants, of the porcine iGb3 synthase gene.
It is another object of this invention to provide novel nucleic acid and amino acid sequences that encode the porcine iGb3 synthase protein.
It is yet a further object of the present invention to provide cells, tissues and/or organs deficient in the porcine iGb3 synthase gene.
It is another object of the present invention to generate animals, particularly pigs, lacking a functional porcine iGb3 synthase gene.
It is yet a further object of the present invention to provide cells, tissues and/or organs deficient in functional porcine iGb3 synthase gene for use in xeontransplantation of non-human organs to human recipients in need thereof.